Neuropeptides are formed from precursor molecules by specific cleavage of peptide bonds catalyzed by endopeptidases. Endopeptidases also participate in neuropeptide degradation. We have isolated two new endopeptidases from brain and pituitary which can generate and inactivate neuropeptides. The enzymes are maximally active at a neutral pH. Rcent experiments in our laboratory have led to the discovery that one of these enzymes, a high molecular weight cation-sensitive neutral endopeptidase can generate methionine enkephalin from Alpha-endorphin and leucine enkephalin from synthetic precursor molecules. This endopeptidase may participate in enkephalin formation in vivo. The enzyme is inhibited by low concentrations of Na+ and K+ implying a role for these cations in the regulation of its activity. The second enzyme, prolyl endopeptidase, highly purified from rabbit brain, hydrolyzes peptiprolyl peptide and peptidyl prolyl amino acid bonds in model substrates and in neuropeptides such as substance, P, neurotensin, thyrotropin-releasing hormone, lutenizing hormone releasing hormone, bradykinin and angiotensin II. The role of these enzymes in the control of the formation and inactivation of neuropeptides will be investigated. The biochemical properties of the two endopeptidases will be studied in greater detail and the relationship of enzyme structure to catalytic properties will be explored. An attempt will be made to localize the enzymes in brain and pituitary by immunocytochemical techniques. Their localization will be correlated to the known distribution of neuropeptides. Active-site directed peptide aldehyde derivatives will be synthesized as potential inhibitors of these enzymes. Such inhibitors may repreesent a new generation of psychotropic agents. Cation-sensitive neutral endopeptidase may be subject to pharmacological or physiological regulation. The effect of morphine treatment and of immobilization stress on the activity of this enkephalin synthetase will be studied. Other neutral endopeptidases of brain and pituitary that we have detected will be isolated and characterized.